Antibiotic-resistance complicates the majority of Staphylococcus aureus (S. aureus) infections, as a full two thirds of hospital associated S. aureus infections and ~50% of those acquired in the community are now methicillin-resistant (MRSA). The increasing incidence of multidrug-resistance in S. aureus and other bacteria underscores the need for next generation antibiotics capable of combating these dangerous pathogens. Ideally, new drugs will not only efficaciously treat contemporary resistant strains, but they will also delay the development of new resistance phenotypes. To do so, new drugs will have to function by mechanisms orthogonal to that of conventional, inhibitory antibiotics. The human immune system has evolved a formidable arsenal of bactericidal agents, and many of these attack bacterial cell structures with less inherent plasticity than the proteins and ribonucleic acids targeted by conventional inhibitory drugs. One example is the human enzyme lysozyme (hLYS), which kills Gram-positive pathogens in part by hydrolysis of cell wall peptidoglycan and subsequent bacterial lysis. While all pathogenic bacteria rely on peptidoglycan for structural stability, some strains are able to evade destruction by hLYS. One common mechanism of hLYS-resistance is subtle structural modifications to cell wall peptidoglycan. In particular, O-acetylation can abrogate hLYS activity, and is a modification known to exist in at least 39 different bacteria including pathogenic Staphylococci and Streptococci. Importantly, the hLYS-resistance of S. aureus can be attributed solely to O-acetylation of peptidoglycan. In this proposal, combinatorial protein engineering will be used to develop entirely novel hLYS variants capable of efficiently degrading the O-acetylated peptidoglycan of S. aureus, thus killing the pathogen. Specific Aim 1 will focus on optimizing an innovative ultra-high throughput antibiotic screen. Recombinant yeast secreting prospective antibacterial proteins are coencapsulated with bacterial targets in 50 5m hydrogel microdroplets (GMDs). GMDs in which the secreted protein kills the bacterial target are fluorescently tagged with a live/dead stain, and are subsequently isolated using high speed fluorescence activated cell sorting (FACS). The GMD-FACS assay has been demonstrated in proof-of-concept experiments, and will be fully optimized using artificial enrichments of positive control cells from large excesses of negative controls. Specific Aim 2 will test the hypothesis that hLYS can be engineered to efficiently hydrolyze O-acetylated peptidoglycan and lyse S. aureus. Leveraging the GMD-FACS assay from aim 1, large combinatorial libraries of mutated hLYS enzymes will be screened for anti-S. Aureus lytic activity. Isolated enzyme variants will be quantitatively characterized with respect to antibiotic potency. Successfully achieving the project objectives will yield a high throughput screen with broad utility in developing antibacterial proteins and peptides, and will also produce entirely novel, therapeutic, human enzymes capable of efficiently killing drug-resistant S. aureus pathogens. PUBLIC HEALTH RELEVANCE: Bacterial pathogens typically begin to develop resistance towards conventional antibiotics shortly after their first therapeutic application. Drug-resistant bacteria, such as MRSA, are becoming increasingly common, and they can transform a common infection into a life threatening illness. This proposal seeks to develop powerful, new, antibacterial enzymes whose therapeutic properties cannot be undermined easily. These next generation therapeutic agents could rearm physicians in the battle against drug-resistant bacterial infections.